Genetic characterization of porcine circovirus 2 found in Malaysia.

BACKGROUND: Porcine circovirus type 2 is the primary etiological agent associated with a group of complex multi-factorial diseases classified as Porcine Circovirus Associated Diseases (PCVAD). Sporadic cases reported in Malaysia in 2007 caused major economic losses to the 2.2 billion Malaysian ringgit (MYR) (approximately 0.7 billion US dollar) swine industry. The objective of the present study was to determine the association between the presence of PCV2 and occurrences of PCVAD. RESULTS: This study showed that 37 out of 42 farms sampled were positive for PCV2 using PCR screening. Thirteen whole genome of PCV2 isolates from pigs with typical PCVAD symptoms were successfully sequenced. These isolates shared 98.3-99.2% similarities with sequences of isolates from The Netherlands. All thirteen isolates fell into the same clade as PCV2b isolates from other countries. Amino acid sequence analysis of the putative capsid protein (ORF2) of the PCV2 revealed that there are three clusters found in Malaysia, namely cluster 1C and 1A/1B. Of interest, three of the isolates (isolates Mal 005, Mal 006 and Mal 010) had a proline substitution for arginine or isoleucine encoded at nt. position 88-89. Eight of the isolates had mutations at the C terminus of the putative capsid protein suggestive of higher pathogenicity which may account for the high reports of PCVAD clinical symptoms in 2007. CONCLUSION: Phylogenetic study suggests that there may be a link between movements of animals by import of breeders into the country being the route of entry of the virus. While it is not possible to eradicate the virus from commercial pigs, the swine industry in Malaysia can be safeguarded by control measures implemented throughout the country. These measures should include improved biosecurity, disease surveillance; vaccination as well as enforcement of regulations formulated to control and prevent the spread of this disease on a national scale.

Commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus.

The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porcine circovirus type 2 (PCV2) genome supplemented in feed could transmit PCV2 to pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Twenty-three PRRSV-free pigs, non-viraemic for PCV2, were housed in bio-safety level 3 facilities and assigned to four groups in a 2×2 factorial design consisting of PRRSV challenge and a negative control. The diet contained 0 or 8kg SDPP per 100kg of feed. PRRSV challenge groups were inoculated intranasally with 2mL of a suspension containing 10(6) TCID(50)/mL PRRSV. The SDPP used in the study contained 7.56×10(5) PCV2 genome copies per gram. Dietary treatments were fed from 4days prior to PRRSV inoculation until 28days post-inoculation (PI). All challenged pigs developed PRRSV viraemia by day 3PI and PRRSV antibodies were detected in sera by day 14PI, with no difference between diet treatments. Neither PRRSV viraemia nor seroconversion was observed in non-challenged pigs. PCV2 was not detected in the serum of any pigs throughout the experimental period. SDPP containing the PCV2 genome supplemented in feed did not result in PCV2 transmission to either healthy or PRRSV-infected pigs under these experimental conditions.

High prevalence of porcine circovirus viremia in newborn piglets in five clinically normal swine breeding herds in North America.

Porcine circovirus type 2 (PCV2) can be vertically transmitted resulting in fetal infection with or without clinical signs and lesions. The primary objective of this study was to assess the prevalence of intrauterine PCV2 infection in clinically normal newborn piglets in conventional pork production facilities. Five commercial breeding herds located in the U.S. and Mexico were included in the study. A total of 125 sows and 3-5 neonatal piglets per sow were arbitrarily selected. Blood and colostrum samples were collected from sows. Blood was collected from piglets prior to suckling. All samples were analyzed for the presence of anti-PCV2 IgG antibodies and presence and amount of PCV2 DNA. In addition, PCV2 DNA positive samples were further subtyped into PCV2a and PCV2b. All (125/125) sow colostrum samples and 96.8% (121/125) of the sow serum samples and 21.4% (107/499) of the piglet pre-suckle serum samples were positive for anti-PCV2 IgG antibody. The overall PCV2 DNA prevalence was 47.2% (59/125) in sow serum, 40.8% (51/125) in sow colostrum, and 39.9% (199/499) in pre-suckle piglet serum. In the PCV2 DNA positive samples, PCV2b was detected at a higher frequency (69.5% for sow serum, 84.3% for sow colostrum, and 74.4% for piglet serum) compared to PCV2a (18.6% for sow serum, 9.8% for sow colostrum, and 15.6% for piglet serum). Concurrent PCV2a and PCV2b infection was detected in 11.9% of the sow serum, in 5.9% of the colostrum samples, and in 10.0% of the piglet serum samples. In conclusion, an unexpectedly high prevalence of PCV2 viremia was detected in healthy sows (serum and colostrum) and their pre-suckle piglets in the five breeding herds investigated and PCV2b was more prevalent than PCV2a. This information adds to the knowledge of PCV2 infection in breeding herds.

Colostral transmission of porcine circovirus 2 (PCV-2): reproduction of post-weaning multisystemic wasting syndrome in pigs fed milk from PCV-2-infected sows with post-natal porcine parvovirus infection or immunostimulation.

Post-weaning multisystemic wasting syndrome (PMWS) was reproduced in pigs fed colostrum and milk from porcine circovirus 2 (PCV-2)-infected sows and infected post-natally with porcine parvovirus (PPV) or immunostimulated. Pregnant sows were inoculated intranasally with either PCV-2 (n=5) or PCV-2-free PK-15 cell lysates (control, n=10) 3 weeks before the expected farrowing date. Newborn piglets from five of the control sows were introduced to PCV-2-infected sows (n=6 for each sow) and allowed to feed on the colostrum for 12 h and then given 15 ml milk five times a day for 7 days. Newborn piglets from the other five control sows were fed colostrum and milk from their own sows. After 7 days, two piglets from each group were randomly selected to confirm PCV-2 infection. Twenty-one pigs fed by PCV-2-infected sows were randomly divided into three groups and subjected to post-natal PPV infection (group 1), immunostimulation (group 2) or no post-natal treatment (group 3). Twenty-one pigs fed by uninfected sows were also randomly divided and subjected to post-natal PCV-2 and PPV infection (group 4), post-natal PCV-2 infection (group 5) or no treatment (group 6, negative control). Body weight was significantly greater in group 6 than in groups 1, 2 and 4 at 49, 52, 56, 59 and 63 days of age. The typical granulomatous inflammatory reaction and lymphoid depletion of PMWS was observed in the lymph nodes of groups 1, 2 and 4 at 63 days of age. Group 3 had significantly fewer PCV-2-positive cells than groups 1, 2 and 4. In conclusion, PCV-2 shed from colostrum and milk is infectious and reproduces PMWS with post-natal PPV infection or immune stimulation.

Influence of husbandry and control measures on porcine circovirus type 2 (PCV-2) dynamics within a farrow-to-finish pig farm: a modelling approach.

We assessed, using a modelling approach, the influence of several management practices within a farrow-to-finish farm on the age of PCV-2 infection. The impact of PCV-2 vaccination with different vaccination schemes on infection dynamics, was also tested. A stochastic individual-based model describing the population dynamics in a typical French farrow-to-finish pig farm was built and coupled with an epidemiological model of PCV-2 infection. The parameters of the infectious model were mainly obtained from previous transmission experiments. Results were subjected to a survival analysis of time-to-infection. For each comparison, the reference situation was no vaccination followed by random mixing of piglets after birth and after weaning. The risk of early infection was significantly reduced when mixing of piglets was reduced at different stages (avoiding cross-fostering and grouping piglets by litters in small pens after weaning, hazard ratio (HR)=0.52 [0.46; 0.59]). Sow-targeted vaccination delayed the infectious process until the waning of passive immunity and piglet-targeted vaccination considerably decreased the force of infection leading to a dramatic decrease of the total number of infections (HR=0.44 [0.37; 0.54]). The effect was even more pronounced when strict management measures were applied (HR=0.24 [0.19; 0.31]). Changing from a low (3%) prevalence of PCV-2-infected semen to a higher one (18%) significantly increased the risk of early infections (HR=1.36 [1.2; 1.53]), whereas reducing replacement rate or changing sow housing from individual crates to group housing had a limited impact on PCV-2 dynamics.

Transmission of different variants of PCV2 and viral dynamics in a research facility with pigs mingled from PMWS-affected herds and non-affected herds.

Post-weaning Multisystemic Wasting Syndrome (PMWS) has been identified in most swine-producing countries worldwide. The disease has resulted in significant health challenges and economic damage to the swine industry. The aim of this study was to determine horizontal transmission of porcine circovirus type 2 (PCV2) and to examine viral dynamics in pigs in a controlled PMWS transmission study. In the study pigs from PMWS-affected herds and non-affected herds were permitted to have close contact (same pen), nose-to-nose contact (to pigs in neighbouring pens) or no physical contact (pen across the aisle and pens in other compartments). By DNA sequence analysis, eight variants of genotype PCV-2b were identified in the research facility. From the spread of these PCV2-variants it was concluded that PCV2 primarily infects through close contact and nose-to-nose contact. PCV2 genome sequences were obtained from selected pigs at arrival to the research facility and again when the same pigs developed PMWS. This analysis showed that pigs from PMWS-affected herds developed PMWS caused by the same variant of PCV2 as they carried when entering the research facility. In contrast, pigs from non-affected herds developed PMWS with PCV2-variants identified in pigs from PMWS-affected herds. This was probably connected to at least 10(3) higher mean serum-titer of PCV2 in pigs from PMWS-affected herds as compared to pigs from non-affected herds at the beginning of the transmission study. The study further showed that pigs able to control the PCV2 infection, as measured by the PCV2-titer in serum, recovered clinically (pigs from PMWS-affected herds) or stayed healthy (pigs from non-affected herds). Like this, pigs with a PCV2 titer below 5x10(8) copies/ml serum during the study period had a chance of recover from the PCV2 infection whereas pigs with PCV2 titers above 5x10(8) copies/ml serum at any time point generally died from PMWS.

Induction of porcine post-weaning multisystemic wasting syndrome (PMWS) in pigs from PMWS unaffected herds following mingling with pigs from PMWS-affected herds.

In this paper we present the results from two experimental studies (I and II) investigating whether post-weaning multisystemic wasting syndrome (PMWS) can be induced in pigs from PMWS unaffected herds by mingling with pigs from PMWS-affected herds and to observe whether transportation and/or mingling of healthy pigs from unaffected herds could induce PMWS. The studies comprised pigs from 12 different herds. Eight herds had PMWS while four were unaffected. All 12 herds were found to be infected with PCV2. Pigs from PMWS-affected herds were mingled with pigs from unaffected herds in four separate compartments in both study I and study II. In addition, in study II, four groups of pigs from unaffected herds were included. Two groups with pigs transported and mingled from unaffected herds and two groups with pigs which were only transported. The PMWS diagnoses on the individual pigs were based on lymphoid depletion, histiocytic proliferation and the presence of giant cells or inclusion bodies together with the demonstration of PCV2 in lymphoid tissue. Healthy pigs, in both studies, developed PMWS 4-5 weeks after mingling with pigs clinically affected with PMWS. None of the pigs from unaffected herds which had no contact with pigs from PMWS-affected herds developed clinical signs of PMWS. Transportation and mingling of pigs from PMWS unaffected herds in combination or alone was insufficient to provoke PMWS.

Porcine circovirus type 2 in muscle and bone marrow is infectious and transmissible to naïve pigs by oral consumption.

Pork products are a possible source of introduction of PCV2 isolates into a pig population. However, limited work has been done on the transmission through meat of porcine circovirus type 2 (PCV2), a virus associated with several disease syndromes in pigs. The objectives of this study were to determine if pork products from PCV2-infected pigs contain PCV2 DNA/antigen and to determine if the PCV2 present in the tissues is infectious by performing in vitro and in vivo studies. Skeletal muscle, bone marrow, and lymphoid tissues from pigs experimentally inoculated with PCV2 were collected 14 days post-inoculation (DPI). The tissues were tested for presence of PCV2 DNA by quantitative real-time PCR, for PCV2 antigen by immunohistochemistry (IHC), and for presence of infectious PCV2 by virus isolation and inoculation of PCV2 naïve pigs. Lymphoid tissues contained the highest amount of PCV2 (positive by PCR, IHC, and virus isolation), bone marrow contained a lower amount of PCV2 (positive by PCR and IHC but negative by virus isolation), and skeletal muscle contained the lowest amount of PCV2 (positive by PCR but negative by IHC and virus isolation). Naïve pigs fed for three consecutive days with either skeletal muscle, bone marrow, or lymphoid tissues all became PCV2 viremic as determined by quantitative real-time PCR on serum starting at 7 DPI. The pigs also seroconverted to PCV2 as determined by PCV2 IgM and IgG ELISA. In addition, PCV2 antigen was detected by IHC stains in lymphoid tissues and intestines collected from the majority of these pigs. Results from this study indicate that uncooked PCV2 DNA positive lymphoid tissues, bone marrow, and skeletal muscle from PCV2 viremic pigs contain sufficient amount of infectious PCV2 to infect naïve pigs by the oral route.

Detection and genetic characterization of porcine circovirus 2 isolates from the first cases of postweaning multisystemic and wasting syndrome in wild boars in Greece.

In 2002, postweaning multisystemic wasting syndrome (PMWS) was diagnosed in a European female wild boar (Sus scrofa), based on the detection of porcine circovirus 2 (PCV2) DNA in various organs, including the uterus, and on histopathologic lesions. This is the first detection of PCV2 DNA in the uterus of a wild boar. Three years later (2005), a wild boar < 6-8 mo of age was found moribund. It presented wasting and dyspnea and finally died. PCV2 DNA was detected in tissue samples, and histopathologic lesions consistent with PMWS were observed. Both wild boars were from neighboring hunting areas in central Greece. Two PCV2 strains from the wild boars were genetically characterized and compared to other reported PCV2 sequences from wild boars and domestic pigs. The PCV-2 sequences from the wild boars in this study were closely related to each other and were grouped with two isolates from wild boars from Hungary. The phylogenetic analysis revealed that the virus might be transmitted between hunting areas. In addition, PCV2 may spread from domestic pigs to wild boars and vice versa.

Post-weaning multisystemic wasting syndrome and other PCV2-related problems in pigs: a 12-year experience.

Post-weaning multisystemic wasting syndrome (PMWS) and other porcine circovirus type 2 (PCV2)-related diseases have been reported throughout the world for about 10 years. The present paper reviews the knowledge acquired in different fields and is largely based on the authors' experience. The horizontal transmission of PCV2 is widely documented. Contact between pigs is the main route of transmission for both PCV2 and PMWS. However, experimental inoculation of PCV2 to pigs does not give consistent results and severe clinical signs as encountered in the field are rarely obtained. It is thus acknowledged that additional conditions are required for the disease to be severe in growing pigs. These are not all known but co-infections are thought to act as triggers. The spread of such triggers/enhancers, which may or may not be infectious, could have played a role in PMWS dissemination via normal national and international trade, in some cases conferring an epidemic pattern to this spread. Most of the risk factors identified in surveys relate to poor biosecurity and inadequate hygiene/husbandry/herd management. The good correlation between viral burden in the tissues and disease severity emphasized the role of infection pressure. Genomic analysis showed great similarities between PCV2 isolates. However, although two main genotypes (genogroups) could be distinguished from the phylogenic trees, and changes with time, no clear relationship with strain virulence was apparent. Isolates detected in PMWS-positive pigs could also be detected in healthy pigs from healthy farms. A strong sow effect was observed in disease expression in the offspring. Colostrum composition and colostrum intake are supposed to be key components of disease expression. Medication is relatively inefficient as a control measure. Commercial PCV2 vaccines are now becoming available. However, losses as a result of PMWS and PCV2-related diseases are greatly reduced by applying appropriate hygiene and husbandry practices.

Sow porcine circovirus type 2 (PCV2) status effect on litter mortality in postweaning multisystemic wasting syndrome (PMWS).

Previous studies have described a "litter effect" associated with mortality in postweaning multisystemic wasting syndrome (PMWS) affected farms. The main objective of this study was to evaluate litter mortality in different PMWS affected farms and to characterize it in relation to three variables of the sow: parity, porcine circovirus type 2 (PCV2) infectious status and PCV2 antibody titres. The study was performed in seven farms that experienced PMWS in nurseries and/or fattening areas. Fifteen sows from each farm were randomly selected from the same farrowing batch. Serum samples were analyzed for antibodies to PCV2 and for genomic detection of PCV2. Four piglets from each sow (60 piglets per farm) were selected and ear-tagged at birth. Out of 420 initial piglets, 104 (25%) died. Sixty three of them (60%) were necropsied, and 40 (63%) diagnosed as PMWS based on case definition criteria. Our results show that sow PCV2 viremia was significantly related to piglet mortality since more piglets per litter died from viremic than from non-viremic sows. Additionally, a significantly greater proportion of animals died from sows that had low antibody titres against PCV2 (39% vs. 18% from sows with medium to high antibody titres). The present study, of exploratory nature, confirms previous results and further characterizes the so called "litter effect" by establishing that the sow PCV2 status had a significant effect on litter mortality in PMWS affected farms.

Postweaning multisystemic wasting syndrome (PMWS) in Sweden from an exotic to an endemic disease.

Postweaning multisystemic wasting syndrome (PMWS) is causally associated with porcine circovirus type 2 (PCV2) infection of pigs. PCV2 was first demonstrated in Swedish pigs in 1993, although the virus was almost certainly present in pigs in the country before that. Despite this, no signs of PMWS were observed in pigs of Sweden until the first outbreak was reported in 2003. The accumulated number of PMWS-affected herds have increased via 16 (2004) and 41 (2005) to 123 in December 2006. Of these herds, 30 (25%) have now been declared free from PMWS. However, a number of other herds have had individual pigs that have fulfilled the demands for PMWS at necropsy and 52 of these herds have been declared negative on herd basis after treatment for intestinal or respiratory diseases, and/or by correcting shortcomings in management of the herd including feed. Thus, individual cases of the disease have been observed in around 200 herds by the end of 2006 and PMWS is now regarded as an endemic disease in Sweden. The pig population of Sweden is geographically isolated, the density of pigs and the pathogen load in the country is low and the use of growth promoters (low dose antibiotics in feed) was prohibited in 1986. Additionally, the trade of animals in Sweden is organised in a restricted way. Because of these factors it is possible to conduct meaningful real-time studies on the transformation of PMWS in Sweden from being an exotic to an endemic disease in a three year time scale. Initially the PMWS cases were concentrated in the southern part of Sweden, but have gradually spread north. The PMWS-positive herds have, in general, had an effective production, but some management errors have constantly been observed in affected herds. Physical links between affected herds are often missing, and the data generated to date on the PMWS outbreaks in Sweden do not suggest an introduction of a new contagious microbe into the country that is responsible for the PMWS outbreaks, nor does PMWS appear to be spread via semen. In Sweden, intensity in rearing, disease preventing measures and immaturity of the piglets appear to be important as predisposing factors to PMWS and, as such, are discussed in this article.